PROJECT SUMMARY Protein homeostasis (proteostasis) and reduction-oxidation (redox) balance are two tightly regulated and mutually associated molecular events. They play crucial roles in many physiological/pathological conditions including cancer. Disruption of proteostasis and redox balance is an effective approach to selectively kill cancer cells. For examples, proteasome inhibitors such as Bortezomib (Velcade) are highly effective in treating numerous cancers, and autophagy inhibitors are being actively pursued as anti-cancer therapeutics. Many clinically effective chemotherapeutic agents such as arsenic trioxide can induce oxidative burst and cell death, which is believed to contribute, at least in part, to their anti-cancer effectiveness. On the other hand, dysregulated proteostasis and redox homeostasis can contribute to oncogenesis by activating numerous pro- survival/growth signaling pathways. The seemingly paradoxical effects (pro- and anti-cancer) are generally thought to be accounted for by the intensity and duration of the stresses, although the precise underlying mechanisms remain largely elusive. The ubiquitin-binding protein, p62 (SQSTM1), among its numerous functions, critically regulates both proteostasis and redox balance, by sequestering certain proteins in aggregates and delivering them to autophagosomes for degradation. This sequestration function of p62 relies on its dimmerization via the hydrogen bond between lysine (K)7 and aspartate (D)69 residues. We recently reported that TRIM21 (Tripartite motif-containing protein 21), a RING domain-containing ubiquitin E3 ligase, directly interacts with and ubiquitylates p62 at K7 via K63-linkage, which abolishes the K7-D69 hydrogen bond and inhibits p62 oligomerization, aggregation, and sequestration functions. One of the client proteins sequestered by p62 is Keap1, a negative regulator of the antioxidant response that suppresses the antioxidant transcription factor Nrf2. TRIM21-mediated p62 K7 ubiquitylation leads to the failure of Keap1 sequestration and suppressed antioxidant response. Conversely, TRIM21-deficient cells display increased p62 oligomerization, protein aggregation, Keap1 sequestration, Nrf2 activation, and antioxidant response. In this project, we propose to study the hypothesis that TRIM21 functions as a stress-adaptation molecule and plays a crucial role in proteostasis and redox homeostasis, by ubiquitylating p62 and negatively regulating its sequestration function, for the underlying mechanisms and biological significance, with a main focus on anti- cancer therapy and oncogenesis. As TRIM21 expression is dysregulated and correlates with prognosis in numerous cancers, accomplishing this project will uncover TRIM21 as a new important regulator for cellular proteostasis and redox homeostasis, and will help reveal the role of TRIM21 in cancer development and therapy.